Oligonucleotides are important materials for research, diagnostic, therapeutic and other purposes. An ever-growing demand for improved oligonucleotides, oligonucleotide analogs and for methods for their preparation and use has arisen.
Modified oligonucleotides are of particular value in molecular biological applications. For example, modified oligonucleotides (e.g., oligonucleotides comprising moieties that are detectable, or that confer altered hybridization properties), find use in diverse areas, e.g., as aids in molecular biology, as pharmaceutical compounds, or as diagnostic agents. Modified oligonucleotides that can block RNA translation are useful as inhibitors of gene expression (e.g., as antisense oligonucleotides, small interfering RNAs (siRNAs), microRNAs (miRNAs), ribozymes, sense oligonucleotides and triplex-forming oligonucleotides).
The discovery of the phosphoramidite method for automated synthesis of natural and modified DNA molecules (Letsinger and Lunsdorf (1976) J. Am. Chem. Soc. 98:3655-3661; Caruthers et al., (1987) Methods Enzymol. 154:287-313; Beaucage and Iyer (1992) Tetrahedron, 48: 2223-2311; Protocols for Oligonucleotides and Analogs. Methods in Molecular Biology, Vol 20, Edited by Sudhir Agraval, Humana Press 1993) has stimulated the development of numerous reagents and methods to introduce specific modifications or functional groups at selected positions within synthesized oligonucleotides (Guzayev et al., (1995) Tetrahedron 51, 9375-9384; Matray et al., (1997) Bioconjugate Chem. 8:99-102; Lyttle et al., (1997) Bioconjugate Chem. 8:193-198).
In some applications it is desirable to label or tag oligonucleotides, (e.g., attach a dye or another functional moiety). Some phosphoramidite reagents comprising tags or labels are commercially available. However, escalating interest in the use of modified synthetic oligonucleotides in the disciplines of biology, medicine, and biotechnology (Agraval and Iyer (1999) Curr. Opin. Biotechnol. 6:12-19; Delivery Strategies for Antisense Oligonucleotide Therapeutics. Ed. Saghir Akhtar, CRC Press, 1995; Matysiak et al., (1997) Nucleosides & Nucleotides 16:855-861; Zhao et al., (2001) Nucleic Acids Res. 29:955-959) has intensified the need for less expensive and more broadly applicable labeling reagents. Some efforts have been focused on the development of strategies for the preparation of oligonucleotides that can be conjugated with other molecules and biological moieties, or attached to solid surfaces post-synthetically (i.e., after synthesis of the oligonucleotide). Some strategies for post-synthetic labeling have made use of synthesis reagents that produce oligonucleotides comprising one or more reactive amine groups or reactive thiol groups. While these reagents are useful in many applications, there remains a need for efficient and cost effective compositions and methods for modifying and conjugating oligonucleotides.